Temporal localization of immunoreactive transforming growth factor beta1 in normal equine skin and in full-thickness dermal wounds

OBJECTIVE: To describe the localization of immunoreactive transforming growth factor (TGF)-beta1 in both normal skin and full-thickness dermal wounds of the limb and the thorax of the horse. STUDY DESIGN: Six full-thickness excisional wounds were created on the lateral aspect of one metacarpal region and on the midthoracic area of each horse. Sequentially collected tissue specimens from wound margins were assessed for TGF-beta1 expression by immunohistochemistry. ANIMALS: Four horses (2 to 4 years of age). METHODS: A neutralizing monoclonal anti-human TGF-beta1 antibody was used to detect the spatial expression of TGF-beta1 protein by immunohistochemical localization in biopsies obtained before wounding and at 12 and 24 hours, and 5, 10, and 14 days. RESULTS: No differences in localization of immunoreactive TGF-beta1 were detected between limb and thorax, for either intact skin or wounds. Unwounded epidermis stained moderately for TGF-beta1 protein throughout all layers, whereas the dermis was relatively devoid of immunoreactivity. During the acute stage of repair, migrating epithelium lost its stain, whereas cells of epidermal appendages remained strongly immunoreactive. The epithelium recovered its TGF-beta1 immunoreactivity during wound remodeling, although cells of the stratum corneum remained negative. Macrophages of the inflammatory exudate had positive cytoplasmic staining that diminished with time. Immunoreactivity of granulation tissue fibroblasts was evident early on and increased throughout the repair process. CONCLUSIONS: TGF-beta1 is constitutively expressed in normal, unwounded equine epithelium. Its expression is upregulated within the skin on injury and is associated with the cells involved in wound repair. CLINICAL RELEVANCE: A more precise understanding of the temporal and spatial expression of TGF-beta1 during wound repair in horses should provide the groundwork for possible future manipulations of both normal and aberrant tissue repair.     

Evaluation of plasma alpha-2-macroglobulin and interactions with tumour necrosis factor-alpha in horses with endotoxemic signs.

The electrophoretic position and behavior of the native and activated forms of equine plasma alpha-2-macroglobulin (alpha 2M) were characterized and compared to human alpha 2M by nondenaturing polyacrylamide-gel electrophoresis (PAGE). Plasma alpha 2M was also compared between 6 normal horses and 6 horses with clinical signs of colic and endotoxemia due to volvulus or enteritis. Native and activated forms of alpha 2M were quantified by PAGE and densitometry. Binding of radio-labeled recombinant human tumour necrosis factor-alpha (125I-rhTNF-alpha) to native and activated forms of equine alpha 2M was also evaluated by autoradiography and densitometry of PAGE. Equine plasma alpha 2M migrated as a single band at a position equivalent to native human alpha 2M. Methylamine-reacted equine plasma samples resulted in faster migration of alpha 2M in a similar position to activated human alpha 2M. However, in methylamine-reacted equine plasma, an intermediate alpha 2M band was consistently present between the bands corresponding to native and activated alpha 2M. Amounts of plasma alpha 2M were similar in normal and endotoxemic horses, and remained in the electrophoretically slow or unreacted native form. The vast majority of 125I-rHuTNF-alpha did not bind to alpha 2M or other equine plasma proteins. 125I-rHuTNF-alpha bound weakly to both native and fast methylamine-reacted equine forms of alpha 2M, although binding was better to the activated form. This study indicates that: (1) equine plasma alpha 2M behaves similarly to human alpha 2M on PAGE, (2) plasma alpha 2M of horses can be activated to electrophoretically fast forms, but it is neither activated nor depleted during endotoxemia, and (3) the binding interactions between equine alpha 2M and TNF-alpha are too low to implicate equine alpha 2M as a regulator of TNF-alpha during endotoxemia in horses.     

Cartilage proteoglycans from normal and osteochondrotic porcine joints.

Modern pigs grow fast but are highly susceptible to degenerative joint abnormalities, including osteochondrosis. Normal and osteochondrotic humeri and femurs were obtained from five normal and ten lame adolescent boars to study cartilage proteoglycans. Histological examination of joints indicated a locally-reduced intensity of proteoglycan staining by safranin-O in lesion areas of cartilage. Cartilage proteoglycans extracted with 4.0 M guanidinium chloride were studied using Sepharose 2B gel chromatography. The proteoglycans from severely osteochondrotic joints were less (P less than 0.05) aggregated and contained a greater (P less than 0.05) proportion of smaller monomers than those from normal joints. Loss or damage of core protein, including its hyaluronic acid-binding regions, may account for the greater proportion of small monomers. The results also indicated that the proportion of hyaluronic acid in the total glycosaminoglycan uronic acid fraction, estimated by Sephadex G-200 chromatography and cellulose acetate electrophoresis, was lower (P less than 0.05) for the extracted proteoglycans than for the residual or the whole cartilage proteoglycans in all joints studied.     

Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells

OBJECTIVE: To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. SAMPLE POPULATION: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. PROCEDURE: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen. RESULTS: MSC cultures exposed to TGF-beta1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-beta1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF-beta1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-beta1 led to dose-related increases in area and intensity of type-II collagen immunoreaction. CONCLUSION: Results suggest that TGF-beta1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner.     

Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon

OBJECTIVES: To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons. ANIMALS: 7 horses. PROCEDURE: Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB). RESULTS: Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected byWLB in normal tendon and markedly increased in damaged tendons. CONCLUSIONS AND CLINICAL RELEVANCE: Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs.     

Spatial and temporal expression of types I and II receptors for transforming growth factor beta in normal equine skin and dermal wounds

OBJECTIVE : To describe immunolocalization of TGF-beta receptors (RI and RII) in normal equine skin and in thoracic or limb wounds, healing normally or with exuberant granulation tissue (EGT). STUDY DESIGN : Group A: six wounds on one metacarpus and one midthoracic area. Group B: six wounds on both metacarpi, one of which was bandaged to stimulate EGT. Immunohistochemistry was used to detect RI and RII expression in wound margins. ANIMALS : Eight horses, randomly assigned to one of two study groups. METHODS : Neutralizing polyclonal anti-rabbit RI and RII antibodies were used to detect spatial expression of RI and RII in biopsies obtained before wounding, at 12 and 24 hours, and 5, 10 and 14 days after wounding. RESULTS : RI and RII were co-localized in both unwounded and wounded skin. There were no differences in cell types staining positively between tissues obtained from the limb and the thorax, or from normally healing limb wounds and limb wounds with EGT, at any time. Because of increased cellularity within EGT, staining intensity of limb wounds with 'proud flesh' was greater than limb wounds healing normally, and thoracic wounds, during the proliferative phase of repair. CONCLUSIONS : Strong expression of RI and RII, particularly in limb wounds with EGT, suggested that signalling for stimulation of matrix proteins is in place to contribute to scarring. CLINICAL RELEVANCE : This information may help determine the appropriate time for using receptor antagonists to prevent scarring of limb wounds of horses.     

Effect of gentamicin sulfate and sodium bicarbonate on the synovium of clinically normal equine antebrachiocarpal joints

The effect of gentamicin sulfate, unbuffered and buffered with sodium bicarbonate, on synovial fluid and membrane of clinically normal equine joints was evaluated. Thirty-six adult horses with clinically normal antebrachiocarpal joints were allotted to 6 treatment groups of 6 horses each. One antebrachiocarpal joint in each horse was chosen for treatment. Group-1 horses were given gentamicin (3 ml; 50 mg/ml); group-2 horses were given sodium bicarbonate (3 ml; 1 mEq/ml); group-3 horses were given gentamicin (3 ml; 50 mg/ml) and sodium bicarbonate (3 ml; 1 mEq/ml); group-4 horses were not treated; and horses of groups 5 and 6 were given polyionic physiologic solution (3 and 6 ml, respectively). Synovial fluid specimens were obtained from 5 horses of each group for cytologic analysis at postinjection hours (PIH) 0, 24, 72, and 192 and for pH determination at PIH 0, 0.25, 0.5, 1, 4, 8, 24, 72, and 192. The sixth horse of each group was euthanatized at PIH 24, and the synovial membrane of the treated and contralateral (nontreated) antebrachiocarpal joints was examined macroscopically and microscopically. After intra-articular gentamicin administration, the mean synovial fluid pH was lowest (5.98) at PIH 0.25, but by PIH 8, it was not significantly different from the control value (group-5 horses). When sodium bicarbonate was combined with gentamicin before intra-articular administration, the mean synovial fluid pH was lowest (7.07) at PIH 0.25, but by PIH 1, it was not significantly different from the control value (group-6 horses).(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary observations on expression of transforming growth factors beta1 and beta3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue

OBJECTIVE: To determine whether transforming growth factor (TGF)-beta1 and -beta3 expression differs between equine limb wounds healing normally and those healing with experimentally induced exuberant granulation tissue (EGT). STUDY DESIGN: Six wounds were created on the lateral aspect of both metacarpi of each horse; one forelimb was untreated, and the other was bandaged to stimulate the development of EGT. Sequential wound biopsies allowed comparison of growth factor expression between the two types of wound. ANIMALS: Four horses (2 to 4 years of age; 350 to 420 kg). METHODS: Wounds were assessed grossly, histologically, and by enzyme-linked immunosorbent assay (ELISA) for TGF-beta1 and -beta3 expression at 12 and 24 hours and 2, 5, 10, and 14 days postoperatively. RESULTS: Bandaged wounds developed EGT. In all wounds, TGF-beta1 peaked early and remained elevated at 14 days. Peak TGF-beta1 concentration was higher in wounds with EGT, but not significantly so. Expression of TGF-beta3 differed from TGF-beta1, with peak TGF-beta3 concentrations being delayed. Concentrations of TGF-beta3 were higher in wounds healing normally, but this difference was not significant. CONCLUSIONS: During both normal and exuberant wound repair, the expression of TGF-beta1 occurred earlier than TGF-beta3 expression. Wounds healing with EGT tended to have higher concentrations of fibrogenic TGF-beta1 and lower concentrations of antifibrotic TGF-beta3 than wounds healing normally, although these differences were not statistically significant. CLINICAL RELEVANCE: This study suggests that the production of EGT in bandaged wounds may be related to increased expression of fibrogenic TGF-beta1 and decreased expression of antifibrotic TGF-beta3. Further investigation of the roles of TGF-beta1 and -beta3 may be important in understanding the molecular control of EGT in horses.     

Expression of transforming growth factor beta(1), beta(3), and basic fibroblast growth factor in full-thickness skin wounds of equine limbs and thorax

OBJECTIVE--To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. STUDY DESIGN--Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. ANIMALS--Four 2- to 4-year-old horses. METHODS--Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints. RESULTS--TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds. CONCLUSIONS--Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression. CLINICAL RELEVANCE--The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic.     

Comparison of hematologic values and transforming growth factor-beta and insulin-like growth factor concentrations in platelet concentrates obtained by use of buffy coat and apheresis methods from equine blood

OBJECTIVE: To evaluate the buffy coat and apheresis methods for preparation of platelet concentrates from equine blood by comparing platelet and growth factor concentrations. ANIMALS: 15 mature mixed-breed geldings. PROCEDURE: Whole blood samples were collected and processed by use of a buffy coat or apheresis method to obtain platelet poor and platelet concentrated fractions. The PCV, WBC count, and platelet count were compared among whole blood samples, platelet poor fractions, concentrates obtained by use of the apheresis method (ie, apheresis platelet concentrates), and concentrates obtained by use of the buffy coat method (ie, buffy coat platelet concentrates). Concentrations of transforming growth factor-beta (ie,TGF-beta1 andTGF-beta2) and insulin-like growth factor were compared between buffy coat and apheresis platelet concentrates. RESULTS: Platelet concentrations were 8.9-fold and 5.2-fold greater in buffy coat and apheresis platelet concentrates, respectively, compared with whole blood. Platelet concentrations were 13.1-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta1 concentrations were 2.8-fold and 3.1-fold greater in buffy coat and apheresis platelet concentrates, respectively, and TGF-beta1 concentrations were 10.5-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta2 concentrations were 3.6-fold greater in apheresis platelet concentrates, compared with whole blood. Platelet concentrations correlated with growth factor concentrations across all blood and platelet fractions. White blood cell counts had a significant positive correlation with TGF-beta1 concentration in buffy coat platelet concentrates. CONCLUSIONS AND CLINICAL RELEVANCE: Platelets and TGF-beta1 can be concentrated reliably from equine blood by use of buffy coat or apheresis methods, without modification of the protocols used for humans.     

Coll2-1, Coll2-1NO<Subscript>2</Subscript> and myeloperoxidase concentrations in the synovial fluid of equine tarsocrural joints affected with osteochondrosis

The measurement of biomarkers that reflect cartilage breakdown is a powerful tool for investigating joint damage caused by disease or injury. Particularly in cases of osteochondrosis, synovial concentrations of these biomarkers may reveal the presence of osteoarthritic changes. Coll2-1, Coll2-1 NO₂ and myeloperoxidase have recently been introduced in equine osteoarticular research but comparison between the concentrations of these markers in OCD affected and healthy joints has not been made. Therefore, this study aimed at reporting the synovial concentrations of these biomarkers in joints affected with osteochondral fragments in the tarsocrural joint compared to unaffected joints. Myeloperoxidase and Coll2-1NO₂ revealed to have similar levels between affected joints and controls. However, in contrast to previous studies using C2C the present study demonstrated that synovial levels of Coll2-1 were significantly elevated in tarsocrural joints affected with osteochondrosis. Thus, Coll2-1 may be an earlier marker of cartilage degeneration than other cartilage degradation markers that have been previously used in equine medicine.     

Plasma concentration of transforming growth factor-β1 and hepatic fibrosis in dogs

Liver fibrosis is a morphologic alteration that accompanies chronic liver diseases. Apart from analysis of liver biopsy specimens, there has been no means of diagnosing and evaluating the course of liver fibrosis in the dog. Several plasma markers, including transforming growth factor beta-1 (TGF-β1), are used to indicate liver fibrosis in humans, but none has been validated for use in dogs. There is a significant correlation between the presence and severity of hepatic fibrosis and the plasma concentration of TGF-β1 in humans with hepatic fibrosis and cirrhosis. The feasibility of using TGF-β1 as a marker for hepatic fibrosis in dogs was evaluated by comparing plasma concentrations in 29 healthy dogs and 18 dogs with liver disease. The plasma concentrations of TGF-β1, were 193 to 598 pg/mL in the healthy dogs, 143 to 475 pg/mL in the 7 dogs with mild hepatic fibrosis or none at all, and 427 to 1289 pg/mL in 11 dogs with moderate to severe hepatic fibrosis. The plasma concentrations of TGF-β1 in the dogs with moderate to severe fibrosis differed significantly (P < 0.001) from those in the other 2 groups, whereas the concentrations in the dogs with mild or no fibrosis did not differ significantly from those in the healthy dogs (P > 0.05). It was concluded that TGF-β1 is a potential plasma marker for hepatic fibrosis in dogs.     

The influence of repeated arthrocentesis and exercise on matrix metalloproteinase and tumour necrosis factor alpha activities in normal equine joints

REASONS FOR PERFORMING STUDY: Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) may be useful as biomarkers of joint disease or inflammation. However, activity of both MMPs and TNF-alpha in synovial fluid (SF) may be influenced by nonpathological factors such as arthrocentesis or exercise. OBJECTIVE: To investigate the influence of repeated arthrocentesis and exercise on MMP and TNF-alpha activities in SF from normal equine joints. METHODS: SF was collected from the left metacarpophalangeal, radiocarpal and tarsocrural joints of 16 horses. Eight of these horses were subsequently subjected to an exercise programme on a treadmill and 8 were box-rested as controls. Arthrocentesis was repeated 14, 145, 17 and 24 days after the start of the exercise programme. General MMP and TNF-alpha activities were determined in SF. RESULTS: Repeated arthrocentesis caused a gradual increase but the exercise regimen no significant increase in MMP activity. There was a significant increase in TNF-alpha activity in SF collected from horses 2 h after cessation of the exercise programme. CONCLUSIONS AND POTENTIAL RELEVANCE: When using MMPs as biomarkers for joint disease, at least 14 days should elapse after previous arthrocentesis before subsequent SF collection. Moderate exercise does not increase MMP activity in SF from normal joints and it may be possible to ignore this as a source of error in evaluating MMP activity in diseased joints.     

Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus.

Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.     

Isolation of equine herpesvirus type 1 from a horse with an acute paralytic disease.

A Standardbred mare became paralyzed shortly after showing signs of an upper respiratory infection. The mare was euthanized and equine herpesvirus type 1 was isolated from the brain and spinal cord.     

Distribution of epidermal growth factor receptor (EGFr) in normal and acute peptic-injured equine gastric squamous epithelium.

Growth factors are important in healing and restoration of injured gastrointestinal tissues and, therefore, we characterised temporally the distribution and density of epidermal growth factor receptor (EGFr) in normal and peptic-injured gastric squamous epithelium of horses. Lesions were induced in the equine gastric squamous epithelium using a feed deprivation protocol that results in prolonged increased gastric acidity. Fifteen mature horses, 9 geldings and 6 mares, age 3 to 20 years, were used and divided into 3 groups: Group 1 (n = 5) were subjected to euthanasia for problems unrelated to the gastrointestinal tract and had normal-appearing gastric squamous mucosal epithelium; Groups 2 (n = 5) and 3 (n = 5) had lesions induced in the gastric squamous epithelium by alternating 24 h periods of feed deprivation and ad libitum access to hay, for a total of 48 h and 96 h, respectively. Following lethal injection of a barbiturate, stomachs were removed and fixed by filling with 4- 6 l 10% buffered formalin. Sections were made from normal stomachs and lesions in the gastric squamous epithelium adjacent to the margo plicatus along the right side of the stomach/greater curvature and the lesser curvature. A modified avidin-biotin immunoperoxidase technique was used to stain the formalin-fixed tissue specimens for EGFr. A computerised image analysis system was used to measure area occupied by EGFr (EGFr area) and mean EGFr density in 4 zones within the epithelium extending from the basal cell layers toward the lumen. Measurements were made of epithelium in an erosion bed, at the margin of an ulcer or erosion, and 10-15 mm distant from the lesion margin. Additionally, EGFr area and density were measured in epithelial cells adjacent to capillaries in the epithelium. Intermittent feed deprivation resulted in erosion and ulceration of the gastric squamous epithelium of each horse. Mean EGFr area and density were greatest (P<0.05) in the basal layer of epithelia from all horses, and EGFr staining diminished progressively toward the lumen. Tissues from Group 3 had significantly greater EGFr area in the lesion margin than epithelia from Group 2. EGFr density was less in the epithelia of erosion beds from Groups 2 and 3 compared to normal epithelium, and EGFr area in Group 2 erosion bed epithelia was significantly less than in normal epithelium and epithelia of Group 3. EGFr area in cells adjacent to epithelial capillaries of Group 3 was significantly greater than that of Group 1. Mitotic cell activity was significantly greater in epithelia associated with ulcers and erosions in Groups 2 and 3 compared to normal tissues from Group 1 horses. Staining for EGFr in the glandular mucosa adjacent to squamous epithelium at the margo plicatus was inconsistent and typically faint when present. EGFr distribution in equine gastric squamous epithelium was greatest in regions of greatest cell proliferation, and these areas were in the basal layers of epithelium and immediately adjacent to capillaries. There was evidence that EGFr is induced in peptic-injured equine gastric squamous epithelium. A receptor ligand, EGF or transforming growth factoralpha, may be a factor in healing of gastric squamous mucosal ulcers in horses. Further research should be directed at identifying this ligand and determining its origin in equine gastric mucosa.     

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.     

Metabolic and mitogenic activities of insulin-like growth factor-1 in interleukin-1-conditioned equine cartilage

OBJECTIVE: To determine response of interleukin-1alpha (IL-1alpha)-conditioned equine articular cartilage explants to insulin-like growth factor-1 (IGF-1). Sample Population-Cartilage from the trochlea and condyles of the femur of a clinically normal 4-year-old horse. PROCEDURE: Effects of IGF-1 (0 to 500 ng/ml) after addition of IL-1alpha were evaluated by assessing matrix responses, using a sulfated glycosaminoglycan (GAG) assay, matrix 35SO4 GAG incorporation, and release of GAG. Mitogenic response was assessed by 3H-thymidine incorporation into DNA and fluorometric assay of total DNA concentration. RESULTS: Human recombinant IL-1alpha (40 ng/ml) increased the amount of labeled GAG released and decreased labeled and total GAG remaining in explants, and IL-1alpha decreased mitogenic response. Addition of IGF-1 counteracted effects seen with IL-1alpha alone. In general, IGF-1 decreased total and labeled GAG released into the medium, compared with IL-1alpha-treated explants (positive-control sample). Values for these variables did not differ significantly from those for negative-control explants. A significant increase in total and newly synthesized GAG in the explants at termination of the experiment was observed with 500 ng of IGF-1/ml. Labeled GAG remaining in explants was greater with treatment at 50 ng of IGF-1/ml, compared with treatment with IL-1alpha alone. Concentrations of 200 ng of IGF-1/ml abolished actions of IL-1alpha and restored DNA synthesis to values similar to those of negative-control explants. CONCLUSIONS AND CLINICAL RELEVANCE: IGF-1 at 500 ng/ml was best at overcoming detrimental effects associated with IL-1alpha in in vitro explants. These beneficial effects may be useful in horses with osteoarthritis.